Reduction of experimental diabetic vascular leakage by delivery of angiostatin with a recombinant adeno-associated virus vector.

Purpose To evaluate the efficacy of recombinant adeno-associated virus (rAAV) vector expressing mouse angiostatin (Kringle domains 1 to 4) in reducing retinal vascular leakage in an experimental diabetic rat model. Methods rAAV-angiostatin was delivered by intravitreal injection to the right eyes of Sprague-Dawley rats. As a control, the contralateral eye received an intravitreal injection of rAAV-lacZ. Gene delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Diabetes was induced by intravenous injection of streptozotocin (STZ). Vascular permeability changes were evaluated by extravascular albumin accumulation and leakage of intravenous-injected fluorescein isothiocynate-bovine serum albumin (FITC-BSA). Effects of rAAV-angiostatin on expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), occludin, and phospho-p42/p44 MAP kinase in retina tissue were analyzed by western blotting. Results The rAAV-angiostatin injections led to sustained angiostatin gene expression in retina as confirmed by RT-PCR, and reduced extravascular albumin accumulation in STZ-induced diabetic retina. Further, rAAV-angiostatin significantly decreased intravascularly injected FITC-BSA leakage at 5 days (p=0.001), 10 days (p<0.001), and 15 days (p=0.001) after STZ-induced diabetes, as compared to the control eyes receiving rAAV-lacZ. Expression of VEGF and phosphorylation of p42/p44 MAP kinase in retina was reduced by rAAV-angiostatin at day 1 (p=0.043 for both VEGF and phospho-p42/p44 MAP kinase) after STZ-induced diabetes compared with rAAV-lacZ eyes. rAAV-angiostatin reduced retinal occludin loss at 10 days after STZ-induced diabetes (n=5, p=0.041). There was no significant difference in retinal PEDF expression between eyes injected with rAAV-angiostatin and rAAV-lacZ. Conclusions Intravitreal delivery of rAAV-angiostatin reduces vascular leakage in an STZ-induced diabetic model. This effect is associated with a reduction in the retinal occludin loss induced by diabetes and downregulation of retinal VEGF and phosphor-p42/p44 MAP kinase expression. This gene transfer approach may reduce diabetic macular edema, providing protection in diabetic patients at risk for macular edema.

The present study was designed to examine the transgenic expression of rAAV-angiostatin in the eye and its effect on vascular permeability in the streptozotocin (STZ)-induced diabetic model. Since it has been demonstrated angiostatin can induce the downregulation of VEGF through the blockade of phosphorylation of p42/p44 MAP kinase [9,13,14], we also studied the relationship between rAAV-angiostatin, p42/p44 MAP kinase, occludin, VEGF, and PEDF in this model.

METHODS
Animals: Male Sprague-Dawley (SD) rats (Charles River Laboratories, WilmingtonMA) weighing approximately 200 g on arrival were used in this study. The animals were cared for in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experimental procedures used aseptic sterile techniques and were approved by the Animal Care and Use Committee of the Mackay Memorial Hospital.
Generation of rAAV-angiostatin: cDNA coding for angiostatin was amplified by polymerase chain reaction (PCR) according to a published report [22]. rAAV encoding mouse angiostatin cDNA or lacZ were constructed by using a threeplasmid cotransfection system as described previously [22][23][24]. Titers of rAAV-angiostatin and rAAV-lacZ were determined by dot blot hybridization using angiostatin cDNA and lacZ as probes [25].
Intravitreal injections of rAAV-angiostatin: After being anesthetized, each animal received an intravitreal injection of rAAV-angiostatin (5 µl, 1.5x10 10 viral particles) as described previously [26]. The contralateral eye of each rat was injected with rAAV-lacZ to serve as a control.
Experimental Diabetes: Experimental diabetes was induced three weeks after intravitreal injection of rAAV. Diabetes was induced with a single 60 mg/kg intravenous injection of streptozotocin (Sigma-Aldrich, St. Louis, MO) in 10 mM citrate buffer, pH 4.5. Animals that served as nondiabetic controls received an equivalent amount of citrate buffer alone [21] Twenty-four h later, rats with blood glucose levels higher than 250 mg/dl were deemed diabetic. These diabetic rats received 6-8U NPH insulin (Lilly, Indianapolis, IN) once a week to prevent ketoacidosis. Just before experimentation, blood glucose levels were measured again to confirm diabetic status.
Reverse transcription-polymerase chain reaction: Expression of rAAV-angiostatin in retina was confirmed by RT-PCR according to a protocol described in reference [8]. Each rat eye that was previously injected with rAAV-angiostatin and rAAV-lacZ was enucleated, and chorioretinal tissues were harvested for RT-PCR at 1, 5, 10, and 15 days after induction of experimental diabetes. The cDNA was synthesized using oligo(dT) primer and 200 IU transcriptase (SuperScript II; Life Technologies, Carlsbad, CA) according to the manufacturer's   Animals were made diabetic by a single streptozocin (STZ) injection (65 mg/kg) in 1 mmol/l sodium citrate buffer, pH 4.5. All diabetic animals had higher blood glucose levels and reduced body weight gain compared to age matched, non-diabetic control animals. The eyes previously injected with rAAV-lacZ (lanes B to E) and rAAV-angiostatin (lanes F to I) were enucleated and chorioretinal tissues were harvested for RT-PCR at 1, 5, 10, and 15 days after induction of experimental diabetes. Lane A is the control eye. Lanes B and F are 1 day after diabetes induction. Lanes C and G are 5 days after diabetes induction. Lanes D and H are 10 days after diabetes induction. Lanes E and I are 15 days after diabetes induction. There was no angiostatin gene expression in the control eye (lane A) and eyes injected with rAAV-lacZ (lanes B to E). In the eyes injected with rAAV-angiostatin, angiostatin gene expression was detected (lanes F to I). As an internal control, expression of GAPDH was detected in normal control eye and eyes receiving both rAAVangiostatin and rAAV-lacZ injections (lanes A to I). "M" indicates molecular weight markers.
instructions. PCR amplification was performed with two oligonucleotide primers, 5'-CAG CAA TGC GTG ATC ATG-3' and 5'-TGG AGA TTT TGC CCT CAT AC-3'. As a control, PCR amplification was performed for glyceradehyde-3-phosphate dehydrogenase (GAPDH) with two oligonucleotide primers, 5'-GGA AGG GCT CAT GAC CAC AG-3' and 5'-CCT TTA GTG GGC CCT CGG-3'. To rule out the possibility that gene amplification products were derived from amplification of contaminating angiostatin genomic DNA, we treated the total RNA with RNase free DNase I (Qiagen, Valencia, CA) before RT-PCR. Immunofluorescence assay: Five-µm-thick retinal tissue sections from formalin-fixed, paraffin-embedded blocks were transferred to positively charged slides to be used for staining. Sections were dewaxed in xylene and progressively hydrated [27]. They were then washed three times with PBS and a 1:400 dilution of polyclonal rabbit antihuman albumin antibody (DAKO Diagnostics. Mississauga, ON, Canada) was applied. A fluorescein isothiocyanate (FITC)-conjugated antimouse IgG was used as a secondary antibody. The results were viewed with a fluorescence microscope (Zeiss Axioplan HBO100, Oberkochen, Germany).
Measurement of leakage with intravascular injected FITC-BSA: Retinal vascular leakage was measured using the intravascular injected FITC-BSA as previously described [21,28] with some modifications. After induction of anesthesia, the rats received tail vein injections of 100 mg/kg FITC-bovine serum albumin (FITC-BSA, Sigma-Aldrich). The animals were sacrificed 20 min later, and their eyes were removed, embedded in OCT medium, and snap-frozen in liquid nitrogen. The  plasma was collected and assayed for fluorescence with an SPEX fluorescence spectrophotometer (Molecular Devices, Sunnyvale, CA) based on standard curves of FITC-BSA in normal rat plasma. Frozen retinal sections (6 µm thick) collected every 60 µm were viewed with a Zeiss Axioplan HBO100 fluorescence microscope. Images from six retinal nonvascular areas (200 µm 2 ) in each section were collected. Quantification of FITC-BSA fluorescence intensity was calculated by computer software Q-win (Leica, Wetzlar, Germany) and normalized to plasma fluorescence intensity for each animal.
Cell culture and rAAV-angiostatin infection: To further evaluate whether angiostatin could induce the expression of PEDF in endothelial cells, we infected human umbilical vein endothelial cells (HUVEC-2C, Cascade Biologics, Portland, OR) with rAAV-angiostatin. The HUVEC-2C cells were cultured in medium 200 (Cascade Biologics) containing low serum growth supplement. All cells were supplemented with 1% penicillin-streptomycin and maintained at 37 °C and 5% CO 2 . Confluent cells obtained during the fifth passage were used for rAAV-angiostatin infection. The HUVEC-2C cells were infected by rAAV-angiostatin in Dulbeco's modified Eagle's The retinal FITC-BSA fluorescence intensity in eyes receiving rAAVangiostatin injection was 2.99±0.62 pixels at 5 days, 3.42±0.38 pixels at 10 days and 3.30±0.40 pixels at 15 days after induction of diabetes. The retinal FITC-BSA fluorescence intensity in eyes receiving rAAV-lacZ injection was 3.59±0.31 pixels at 5 days, 3.77±0.51 pixels at 10 days and 3.71±0.47 pixels at 15 days after induction of diabetes. The normalized FITC-BSA fluorescence intensity in eyes receiving rAAV-angiostatin was decreased as compared to eyes receiving rAAV-lacZ at 5 days (t=3.67, n=49, p=0.001), 10 days (t=3.94, n=51, p<0.001), and 15 days (t=3.52, n=56, p=0.001) after STZ-induction of diabetes. The asterisk indicates a p less than or equal to 0.001. Four SD rats were represented by the number of sections (n) to be examined. The rats received intravitreal injection of rAAV-angiostatin in the right eyes and rAAV-lacZ in left eyes, and diabetes was induced three weeks after injection. A: Each blot is a representative of the results from five rats. B: Occludin levels were semi-quantified by densitometry, and normalized to actin. The rAAV-angiostatin significantly decreased retinal occludin loss as compared to eyes receiving rAAV-lacZ injection at 10 days after induction of diabetes (the asterisk indicates significance using the Wilcoxon signed rank test, n=5, p=0.041). 136 medium for two days. Cell lysates were then prepared and analyzed for PEDF by western blotting as described in the previous paragraph.
Statistical Analyses: The results are expressed as the mean±SD. Retinal FITC-BSA fluorescence intensity was analyzed in serial retinal sections from four rats at 5, 10, and 15 days after induction of diabetes. Due to skewed distributions, data were subjected to logarithmic transformation for analysis. Differences of retinal FITC-BSA fluorescence intensity between eyes receiving rAAV-angiostatin and rAAV-lacZ injection at 5, 10, and 15 days after induction of diabetes were analyzed by a paired-sample Student's t test. The retinal expression of VEGF, PEDF, phosporylation of p42/p44 MAPK, and occludin were analyzed by the Wilcoxon signed-rank test. All p-values are two-tailed, and differences were considered to be statistically significant for p<0.05.

RESULTS
Gene delivery by rAAV-angiostatin: There was no angiostatin gene expression in normal control eyes (Figure 1, lane A) and eyes injected with rAAV-lacZ at 1, 5, 10, and 15 days after induction of diabetes (Figure 1, lanes B-E). In the eyes injected with rAAV-angiostatin, angiostatin gene expression was detected at 1, 5, 10, and 15 days after induction of diabetes ( Figure 1, lanes F-I). As an internal control, expression of GAPDH was detected in normal control eye and eyes receiving both rAAV-angiostatin and rAAV-lacZ injections ( Figure  1, lanes A-I).
Induction of experimental diabetes: Animals (n=12) with a blood glucose exceeding 250 mg/dl were selected for inclusion in the diabetic group. All diabetic animals had higher blood glucose levels and reduced body weight gain at 5, 10, and 15 days after induction of diabetes compared to age-matched, nondiabetic control animals ( Table 1).
rAAV-angiostatin influence on vascular permeability: One week after induction of diabetes, the immunofluorescence assay, using anti-albumin antibody, disclosed staining only inside blood vessels in normal control eyes (Figure 2A). In-  There was no apparent difference in retinal PEDF expression between eyes exposed to rAAV-angiostatin and rAAV-lacZ. at 1, 5, 10, or 15 days after STZ injection. B: There was no increase in PEDF expression in HUVEC-2C cells receiving rAAVangiostatin compared to control HUVEC-2C cells. Western blotting of the PEDF plasmid-transfected HUVEC-2C cells was used as a positive control. 137 creased extravascular albumin in the retinal parenchyma was seen in the eyes of diabetic animals receiving rAAV-lacZ (Figure 2B). In the diabetic animals receiving rAAV-angiostatin, however, albumin staining was observed only inside blood vessels ( Figure 2C).
To confirm the effect of rAAV-angiostatin on vascular permeability, we examined animals injected with intravenous FITC-BSA 5, 10, and 15 days after induction of diabetes. Figure 3 shows representative micrographs of the eyes of normal control and STZ-induced diabetic rats. FITC-BSA fluorescence is limited to the vasculature in the normal retina ( Figure 3B) and diffusely increased throughout the retinal parenchyma at 5 days after STZ-induced diabetes in eyes receiving rAAV-lacZ injection ( Figure 3C). Increased fluorescence intensity throughout the retinal parenchyma is still present at 10 (Figure 3D) and 15 ( Figure 3E) days after STZ-induced diabetes in eyes receiving rAAV-lacZ injection. Little fluorescence was present in the retinal parenchyma in eyes receiving rAAVangiostatin injection ( Figure 3F) at 5 days after induction of diabetes. Retinal parenchyma fluorescence at 10 ( Figure 3G) and 15 ( Figure 3H) days after induction of diabetes in eyes with rAAV-angiostatin injection was decreased as compared with eyes that received the rAAV-lacZ injection.
The retinal FITC-BSA fluorescence intensity was calculated and normalized to plasma fluorescence intensity by image analysis of serial sections (Figure 4). Four SD rats were represented by the number of sections to be examined at each timepoint. The mean retinal FITC-BSA fluorescence intensity in eyes receiving rAAV-angiostatin injection was 2.99±0.62 pixels at 5 days, 3.42±0.38 pixels at 10 days, and 3.30±0.40 pixels at 15 days after induction of diabetes. The retinal FITC-BSA fluorescence intensity in eyes receiving rAAV-lacZ injection was 3.59±0.31 pixels at 5 days, 3.77±0.51 pixels at 10 days, and 3.71±0.47 pixels at 15 days after induction of diabetes. Quantitative analysis showed that the fluorescence was decreased in eyes receiving rAAV-angiostatin as compared to eyes receiving rAAV-lacZ at 5 days (t=3.67, n=49, p=0.001), 10 days (t=3.94, n=51, p<0.001), and 15 days (t=3.52, n=56, p=0.001) after induction of diabetes.

DISCUSSION
Retinal vascular leakage is a major cause of macular edema in diabetic retinopathy and other retinal diseases [1][2][3][4][5][6]. Traditionally, laser photocoagulation has been used to reduce the vascular leakage induced by diabetes [32,33]. Recently, antiinflammatory drugs such as triamcinolone [34], or pars-plana vitrectomy with or without internal limiting membrane peeling [35] have been used to attenuate the diabetic macular edema. Due to the duration and severity, an ideal strategy would be to develop an approach involving a single administration of a vector that would result in long-term expression of a suitable therapeutic gene [36]. rAAV vectors are highly efficient gene delivery systems which can facilitate long-term transduction [22,23]. We have previously reported in several animal models a gene transfer technique based on the use of rAAV vectors [8,23,25,26,37,38]. This gene transfer technique is particularly attractive for treating ocular disease for reasons of accessibility and long term transduction, and potentially because it would enable clinicians to avoid repeated intravitreal injection [8,37]. We previously reported suppression of laser-induced choroidal neovascularization by an rAAV vector expressing angiostatin [8]. Here, we report that vascular leakage in experimental diabetic rats can be reduced by angiostatin delivery via an rAAV vector. These results suggest that rAAV-angiostatin could be beneficial in the treatment of diabetic macular edema.
Angiostatin is a proteolytic fragment of plasminogen and contains kringle domains 1 through 4 [10]. It has been determined that angiostatin is a potent anti-angiogenic factor [11] that can inhibit endothelial cell migration and induce apoptosis in these cells [12]. Recently, intravitreal injection of angiostatin was found to reduce vascular leakage in a rat model of experimental diabetes and in oxygen induced retinopathy [9]. In the same report, the expression of VEGF was found to be downregulated by angiostatin.
VEGF is a potent angiogenic factor, expression of which is increased in eyes with diabetic retinopathy [13,14]. VEGF, which is also known as vascular permeability factor (VPF) [39], increases microvascular permeability at very low concentrations [39], and may be important in the pathogenesis of vascular leakage induced by diabetes [40]. Angiostatin-induced reduction of vascular leakage occurs through blockade of VEGF expression [15]. In our study, retinal VEGF expression decreased in eyes receiving rAAV-angiostatin as compared to rAAV-lacZ treated eyes at one and five days after induction of diabetes. Vascular leakage was also decreased in eyes receiving intravitreal injection of rAAV-angiostatin compared to the contralateral eyes receiving rAAV-lacZ injection at 5, 10, and 15 days after induction of diabetes.These results are consistent with previous reports that angiostatin reduces the vascular leakage via blockade of VEGF [13][14][15]39,40]. How angiostatin reduces vascular leakage through inhibition of VEGF production is still under investigation.
It is known that p42/p44 MAP kinase phosphorylation is induced by hypoxia [41]. This phosphorylation is diminished by angiostatin in microvascular endothelial cells [42]. Phosphorylation of p42/p44 MAP kinase promotes VEGF expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex of the VEGF promoter [43]. It is therefore plausible that inhibition of phosphorylation of p42/p44 MAP kinase by angiostatin suppresses VEGF expression under conditions of hypoxia. In our study, retinal phosphorylation of p42/p44 MAP kinase decreased in eyes receiving rAAV-angiostatin at one day after induction of diabetes ( Figure 8). Our results in STZ-induced diabetic rats suggest that angiostatin suppresses VEGF expression by inhibition of phosphorylation of the p42/p44 MAP kinase.
BRB breakdown is a hallmark of vascular leakage in diabetic retinopathy and other retinal vascular diseases [44,45]. The tight junctions between the retinal vascular endothelial cells constitute an essential structured component of BRB [18]. This barrier limits diffusion of molecules from vessel lumen to the tissue, and thereby maintains the microenvironment of the retina [46]. The barrier protein occludin is decreased in experimental diabetes [21]. VEGF stimulates phosphorylation and redistribution of occludin [19], which is subsequently endocytosed and degraded [47,48]. This process is closely related to the elevated vascular permeability in experimental diabetes [18]. In our study, retinal occludin content in STZinduced diabetic rats was preserved in eyes receiving rAAVangiostatin as compared to eyes receiving rAAV-lacZ. The rAAV-angiostatin-induced inhibition of VEGF may therefore suppress vascular leakage by preserving retinal occludin content in STZ-induced diabetic rats PEDF is a potent angiogenic inhibitor that is counter balanced by the angiogenic effect of VEGF [49][50][51]. Decreased expression of PEDF in retina is associated with ischemia-induced retinal neovascularization and proliferative diabetic retinopathy [50]. Recently, another proteolytic fragment of plasminogen-kringle 5 (K5) was noted to upregulate PEDF expression in a dose-dependent manner in vascular endothelial cells and in the retina [15]. In our study, there was no statisti-cally significant difference between PEDF expression in eyes receiving rAAV-angiostatin and the contralateral eyes receiving rAAV-lacZ. Our results also revealed that rAAV-angiostatin did not upregulate PEDF expression in HUVEC-2C cells. However, PEDF expression has been shown to be induced at both the mRNA and protein level following injury in the eye [52]. Further research is warranted to explore the effect of rAAV-angiostatin on PEDF expression.
Our study showed that transgenic expression of rAAVangiostatin can reduce retinal vascular leakage in STZ-induced diabetic rats. This effect is associated with downregulation of retinal VEGF and phospho-p42/p44 MAP kinase expression, and a reduction in the retinal occludin loss induced by diabetes. However, the vascular leakage and VEGF expression after induction of diabetes in the SD rat model was demonstrated to be a short-term effect [53]. Gene-based therapies can be as effective and viable as real treatment if long-term expression can be achieved. To demonstrate the long-term effect of rAAVangiostatin on vascular leakage induced by diabetes, an alternative animal model such as the Brown-Norway rat could be used in a future study.
Diabetic macular edema is a major cause of vision loss in diabetic patients [7]. On the basis of these findings, we believe that a similar vector and therapeutic gene could eventually be a useful strategy for long-term preventive or adjunctive therapy for macular edema induced by diabetes. It could serve as the basis for an alternative treatment for patients who are suffering from diabetic macular edema as well as a potentially preventive therapeutic modality for diabetic patients who are at risk for development of macular edema.